Nucleocidin and the process of obtaining the same



NGV. 24, 1959 s, 0, THOMAS ETAL. 2,914,525

NUCLEOCIDIN AND THE PROCESS OF OBTAINING THE SAME Filed March 18, 1957 @Gv 99m. a NN Dnvm. DGM.

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IN VEN TORS. SAMUEL 0. THOMAS VER N 0N l.. SINGLETU/V JAMES A. LOWE/'7)' 5X my a ATTORNEY NUCLEOCIDN AND THE PRCESS F OBTG THE SAME Samuel Owen Thomas, VPearl River, and .lames Alfred Lowery, New City, NX., and Vernon Le Roy Singieton, Kaneohe, ahn, Hawaii, assignors to American Cyanamid Company, New York, NX., a corporation of Maine Application March 18, 1957, Serial No. `647,179

6 Claims. (Cl. i60-211.5)

includes the antibiotic now called Nucleocidin, its salts,

and methods of producing and isolating the same.

Nucleocidin in its puried state is a white crystalline material having weakly basic properties. One analytical sample prepared as described hereinafter gave the following results on elemental analysis: carbon 34.03-hydrogen 40S-nitrogen 21.60-sulphur S30- oxygen 32.024, by difference. The compound is of relatively low molecular weight. Its empirical formula based on the above analysis corresponds closely to C11H16N6O8S.

The new antibiotic is almost completely stable in Water at room temperature for 24 hours at hydrogen ion concentrations of pH 3, pH 7, and pH 9. At elevated temperatures it shows some instability, Iparticularly at 100 C. and over.

The infrared absorption spectra of the antibiotic is shown in Figure 1. This curve which is a draftmans reproduction of a tracing made by a Perkin-Elmer Automatic Recording Infrared Absorption Spectrophotometer Model 21 was determined on a sample of the material which had been mixed with crystals of KBr and pressed into a disc. The original curve was made on the machine under the following conditions: Prism=NaCl; Resolution=2; Response=1-1; Gain=5; Speed=1/2 min./u; Suppression=2g Scale=2"/u. sorption bands were observed at the quencies expressed in microns:

Significant abfollowing vfre- The ultraviolet absorption spectrum with an aqueous solution containing gammas per milliliter of the antibiotic at pH 7.0 is shown in Figure 2. The maximum absorption peak is at 256 millimicrons and at that value the molecular extinction coecient is 15,500.

The optical rotation of a puriied sample of the antiatent O 2,914,525 Patented Nov. 24, 1959 ice biotic was [a]D24.5=33.3 (1.052 gramsln ml.of 50 percent ethanol-50 percent 0.1 N 'hydrochloric acidi Corrected observed rotation=0.35 in a l1dec.tube)l The new antibiotic is soluble in the following solvents at room temperature at approximately the levels `indicated which are expressed in milligrams per milliliter of solvent:

Water.pH 3.5 p Y Water.pH 6.5 11.9 Water.pH 9.25 126.8 Methanol 1 14;2

Acetone 4.8 n -Butanol 0.25 Ethyl acetate 0.36 Benzene V 0.137

Ethel 1 These gures obtained gravimetrically.

The new antibiotic of the present invention is strongly adsorbed on activated carbonover a'wide range of lhydrogen ion concentrations ranging/from below pH 3 lto v 'I above pH 9. It is' weakly adsorbed on such common adsorbents as activated magnesium silicate, alumina, alpha cellulose, and fullers earth under acidic, neutral and alkaline pH conditions. `It cank be adsorbed on cationic exchange resins from neutral or slightly'alkaline solutions and can be eluted therefrom with an acidic or basic solution. ,l

Degradation studies have has the following structure:

indicated that Nucleocidin i i O Organism: Zone width, mm. B. cereus Waksrnan 8.0 K. pneumoniae (Friedlanders) 2.9 Salmonella gullinczrum pH 7.8 `7.0 `Salmonella gallinarum pH 6.0 6.1 Alcaligenes sp. ATCC10153 sl. Staph. aureus (No. 209) 6.0 B. subtilis pH 7.8 8.9 B. subtilis pH 6.0 9.4 B. subtilis streptothricin resistant 3.0 M. ranae 0 M. tuberculosis pH 7.8 2.9 M. tuberculosis pH 6.0 7.1 Staph. albus 8.0 K. pneumoniae 7.5 E. coli 7.0 Streptococcus haemolyticus NYS 16.1 Corynebacterium xerose '7.0

Trypanosomiasis in animals of economic importance occurs in various parts of the world and is caused by several diiferent species of the protozoan genus Trypanosoma. In Africa, cattle and swine are seriously alected, the principal species being T. congolense, T. vvax and T. simae. Elsewhere, various other species cause serious diseases in equines, camels, sheep and goats. T. equperdum causes a disease known as dourine in equines. 'This occurs in southern Europe, Asia, North and South America, and South Africa.

Because of the economic important of trypanosomisis in many types of livestock, a search for new drugs to treat and-cure the disease has been made. Obviously it is not possible to use large animals for laboratory investigations and work of this kind. Y Fortunately, most species of trypanosomes can be inoculated experimentally into small laboratory animalsV and produce fatal infections comparable to those which occur in the natural hosts. Experimental infections in mice, rats, rabbits, or guinea pigs, using one or several species of trypanosomes, are utilized. A standardized infection in mice with T. equperdum was used in our work since it is invariably fatal unless efective treatment is given.

The validity of this work is demonstrated by the fact that the current drugs of choice for the treatment of cattle trypanosomiasis were rst evaluated in mice and the data was used to project dosage schedules to cattle. With all'compounds now used to treat trypanosomiasis in cattle and horses, the carry-over of activity from the mouse test was 100%.

A comparison of the effectiveness of Nucleocidin against T. equpedum in mice was made against Antrycide sulfate and Suramin sodium. The former being 4-amino-6-(2-amino-6-methyl-4-pyrimdylamino) quinaldine-1,1 dimethosulfate; and the latter Hexasodium sym.- bis (m-amino-p-methylbenzoyl-l-naphthylamino 4,6,8- trisulfonate) carbamide. Mice were given a single intramuscular dose of the drugs as shown in the following table with the results as indicated.

These results indicated that a single parenteral dose, 0.01 to 0.1 ing/kg. of Nucleocidin should be effective in treating cattle and horses with trypanosomiasis In addition to the antitrypanosomal activity of Nucleocidin, it is also eifective against experimental amebiasis in guinea pigs. The protozoan Endamoeba histolytica is the causative agent of amebiasis. Dogs may become infected. Serious symptomsmay develop, and untreated infections may be fatal.` The disease is widespread throughout the World. v

Experimental evalutions of the activity of compounds against this parasite are usually conducted in laboratory animals infected with virulent strains of Ena'amoebav histolytica. Most compounds Arecently introduced for the treatment of amebiasis in dogs were first evaluated lin experimentally-infected guinea pigs or rats, and efective doses were projected to the naturally-infected OSS.

In guinea pigs experimentally infected with Endamoeba histolytica, Nucleocidin has proven eifective in multiple oral doses of 0.125 and 0.25 ing/kg. twice daily for tive days (total dosage=l.25 and 2.5 mg./kg.) and in single oral doses of 1.0 mg./kg. This degree of activity is considerably greater than that of other known amebacides, such as Diodoquin, milibis, Carbarsone and the Tetracyclines.

Nucleocidin is highly toxic to rats and mice when added to their feed and is useful in this regard as a rodenticide.

To a commercially available rat diet was added varying amounts of Nucleocidin and groups of rats were allowed to eat this material adlibitum. The rat diet was composed of the following ingredients: meat meal, dried skimmed milk, wheat germ, iish meal, liver meal, dried beet pulp, corn grits, oat middlings, soybean oil meal, dehydrated alfalfa meal, cane molasses, vitamin B12 supplement, ribotlavin supplement, brewers dried yeast, thiamin, niacin, vitamin A feeding oil, D activated plant sterol, .5% delluorinated phosphate, .5% iodized salt, .02% manganese sulfate.

A concentration of 0.05 percent of Nucleocidin in the feed killed all of the rats allowed access to this feed within three days. From the amount of the rodenticide consumed by the rats, it was estimated that 750 gammas of Nucleocidin per rat was ingested on the first day (about 1.9 mg./kg.) and Z50-500 gammas on the second day (about 0.9 nig/kg). A concentration of Warfarin- 3-7-phenyl--acetylethyl 4 hydroxycoumarin, a Well known rodenticide required to kill rats is 0.1% in the feed over a period of four days.

Nucleocidin is produced by a species ofStreptomyces bearing the ATCC No. 13,382. Further investigation of the taxonomy of this microorganism indicates that it is a previously undescribed species. It has been named Strepomyces calvus (calvus meaning bald or bare) because of its generally poor display of aerial mycelium and sporulation on most media as shown in Table I. On those media which support aerial growth and sporulation, the mycelium, at lrst appears white, later becoming Drab Gray to Light Mouse Gray with the maturation of spores; sporulation was heaviest on starch-containing media (Waksman starch and Czapek starch). The vegetative myceliurn grows appressed, often spreading, and is colorless to Ivory Yellow or even Honey Yellow on some media. The reverse coloration of the colonies varies with the medium from colorless to Ivory Yellow or Honey Yellow and occasionally to Cinnamon-Buf1. Soluble pigment is not produced by the parent culture; however, some induced mutant strains have been observed to produce a limited amount of yelllowish pigment. Although S. calvus will grow at widely divergent temperatures, it was established that the optimum range both for growth and sporulation is 32-37 C.

Spores of S. calvus are borne in short chains which sometimes -form short loose spirals of one to several turns; individual spores are globose to elongate (0.6- 1.0a in globose cells to 0.6-1.0,u x 1.0-1.8M in elongate cells).

One physiologic property of S. calvus, which separates it from most other Streptomyces, is its capacity Vto cause formation of crystalline pellets of indeterminate nature in growth zones on calcium malate agar;` these pellets eiervesce in the presence of acids.

The carbon source utilization pattern of S. calvlls (T-30l8) was determined to be as follows: d-fructose, i-inositol, lactose, d-mannitol, d-raflinose, l-rhamnose, sucrose, d-trehalose and d-xylose are readily utilized; l-arabinose, d-melibiose and salicin are utilized very poorly; dextran, esculin, d-melezitose and adonitol are not utilized at all.

The determinations were made bythe technique of Pridham and Gottlieb: The utilization of carbon compounds by some Actinomycetales as an aid 4for species determination, Jour. Bact. V56,' 107, 1948.

TABLE I Cultural and physiologic characteristics of 'Streptomycesd calvus (Incubation 14 days at 32 C.) `v

` Amount andv Amount and Color of Aerial Color of Vegetative Diiusi- -v Medium Nature of Mycelium and Spores Mycelium Reverse Color ble v Remarks Growth Y Y l Pigment l Asparagine dextrose Meat Moderate--- Scanty; White aerial growth; Colorless to Cream Colorless to Cream NoneJ...

ExtractrAgarvpH 6.543.?. Y No sporulation. Color.l Color. g v Y Asparagiue dextrose Meat Fair4 None Colorless to Yel- .--..do -.-do..,.

Extract agar, pH 5.0. lowish. f Bennett agar Moderate; Scanty; white aerial growth; do Near C M1 .rin

Spreading No sporulation.` Buy?. Calcium malate agar Moderate-.- Scanty; white to Drab Gray in White to Cream Colorless to near do... Wide zone of malate sporulating areas of aerial Ballr to Honey Hoher/Yellow. l clearing; crystalline growth. 'sporulation very y Yellow. pellets formed in light.' growth zones. Corn steep liquor agar..-. Light; Scanty, White aerial growth; Light yellowish Chamoisv to Honey do Spreading, No sporulation. o an. ellow. Czapek-Dox agar Moderate; Scanty; white to Drab Gray in Cream-Baita Chamois-. .do

Spreading. sporulating areas. Chamois. 1 Czapek-Dox Starch agar- ..do- Moderate, White to Light Colorless to Colorless to Light do- .Slight starch `Mouse Gray in sporulating Chamois. Bayr to Cream hydrolysis.

' areas; sporulation light. Color. Emerson agar do Seanty; White to Light Mouse `Light yellowish... Honey Yellow...--

' Gray in sporulating areas;

Sporulation very light. Krainsky dextrose agar". Fair Seanty,V White aerial growth; Ivory Yellow Ivory Yellow to do---.

No sporulation. f Cream-Ba. v Potato dextrose agar Moderate; Soanty;'white to Light lllouse Colorless to Ivory Colorless to Ivory .;do-...

Spreading.` Gray in sporulating areas; Yellow to lYellow to l- Y sporulation very light. Cream-Ba17 CreamfBuF. l Sabouraud maltose agar--. Good; Scanty; white aerial growth; Colorless to Honey Yellow to ...do Spreading. No sporulation. Cream-Ba, Ochraceoas. Waksman dextrose agar.-. Moderate; --.do Colorless to Yel Colorless to do Spreading. lowish. Chamois to v p Honey Yellow. f' Waksman nutrient agar... Moderate... .--.-do Light ye11owish.. Cream-Baliv do l Waksman starch agar-- Moderate; Moderate; white to Drab Gray Oolorless to Colorless tol lo ,Slight starch Spreading. o r Light Mouse Gray in spor- Chamois. Chamois; darkhydrolysis.

ulating areas. sporulation ened areas Drabl fain Gray. Yeast extract agar do Scanty; white toV Drab Gray; Light yellowish-.- Chamois to 'Honey d'o No sporulation. Yellow. y Carrot plugs. Poor- Nonev Clorleiss to Yel- Y owis Potato plugs Moderate... Scnty; white to Light Mouse .Clay C0l0r.....,.. Plug-'discolored rai/. i Gelatin do No11e -`.i CtlJlOrleSs t0 Yel- None- Partial llquefaction.

. Y ow. ff. Purple Milk. Light. .....do Colorless to Yel- ...do- Precipitation of casein l lowish. 'l Y andj moderate vpepllwn; final DH Cellulose (Filter paper inv ....-do ...do. ."..-.f.-. Yellowlsh do.-.. No Decomposition Ozapek Solution). l -K i lafter `21 days.

1 italicized colors are those of Ridgivay (8).

THE FERMENTATION PROCESS Y The process by which the new antibiotic is produced is preferably an aerobic fermentation of an aqueousnu` trient medium inoculated with the new. organism de-' scribed above. The constituents of the fermentation medium and the conditions of the fermentation are generally those of other fermentation processes in which fungi are employed to produce antibiotics.

Sources of carbon include starch, hydrolyzed starch, sugars such as lactose, maltose, dextrose, sucrose, or sugar sources such as molasses; alcohols, such as glycerol and mannitol; organic acid, such as citric acid and acetic acid; and various natural products which may contain in addition to carbonaceous substances various other nur 'crient materials. Nitrogen sources include proteins, such as casein, zein, lactalbumin; protein hydrolyzates, proteoses, peptones, peptides, and commercially available materials, such as N-Z Amine which is understood' to be a casein hydrolyzate; also corn steep liquor, soy bean meal, gluten, cotton seed meal, fish meal, meat extracts, stick liquor, liver cake, yeast extracts, distillers solubles, and the like; amino acids, urea, ammonium and 'nitrate salts, and so forth. Inorganic cations, such as sodium, potassium, calcium, magnesium, and so forth, and anions, such as the chloride, sulfate, phosphate, and various combinations of these anions and cations in the form of mineral salts are advantageously used in the fermentation. The so-called trace elements, such as boron, cobalt, iron, copper, zinc, manganese, chromium, molybdenum, and still others may be used to advantage. Generally, the sulfur of the antibiotic and the trace elements occur in suicient quantities in the carbonaceous and usually at the rate of about l volume of air per volume of yfermentation medium per minute. To minimize con tamination with foreign microorganisms, the fermentation vessels should be closed and a pressure of 2-15 pounds above atmospheric pressure maintained in the vessel. Mechanical agitationl in addition to the agitation provided 'by the aeration is generally advisable. Antifoaming agents, 511611 as 1 percentl octadecanol in lard oil, may be added from time to time as required'to prevent excessive foaming.

Fermentation is conducted at a temperature preferably on the order of 26-30 C. out may be as 10W as 17 C. Q rashsh as 4 2. C.

The l time required for maximum production of antibiotics will vary considerably depending upon other conditions of the fermentation. Generally," about 48 hours is required before appreciable quantities of the antibiotic are detected vin the medium. The production of the antibiotic increases with the time, and the fermentation may run as long as hours. The hydrogen i011 conditions normally vary from about pH 6 -pH 8.0, although deviations from these values are permissible.

EXAMPLE 1 An inoculum suitable for starting a large scale fermentation Was prepared in thefollowing manner.

Five to ten ml. of sterile water is used to suspend the surface growth of an agar test tube slant of the culture. The resulting suspension of spores and bits of mycelium is used to inoculate two 100 ml. lots of sterile medium in 50G-ml. Erlenmeyer flasks. The two flasks, after inoculation, are incubated on a reciprocating shaker at about 28 C. foi-'about 2 days, after which the 200 ml. of primary inoculum is used to inoculate 6 liters of sterile medium 4in a 9-liter glass bottle, which in turn is incubated, with aeration, usually for a day or so at about 28 C. The 6 liters of bottle culture is then used to inoculate 10G-200 liters or more of sterile medium in a fermenter tank. For larger lots, 500 liters or more, larger inocula are ordinarily obtained by combining two or more 6-liter bottle inocula.

A fermentation medium of the following composition:

1D. R. Hoagland and W. C. Snyder appearing in the Proceedings of the American Society for Horticultural Science, volume 30, pages 288-294, 1933. was made up to 1,000 milliliters with water and adjusted to pH 7-7.2 with aqueous sodium hydroxide.

Fifteen hundred liters of this medium was placed in a 500 gallon fermenter tank, sterilized about 60 minutes at l5 pounds steam pressure (120 CJ, and inoculated with l2 liters of 23hoursold bottle culture as described above. The pH was 6.96 before sterilization and 6.68 after sterilization. The mixture vwas fermented for 112.5 hours at 26-290 C. (most of the time 27-28 (2.).

In addition to the above fermentation, a number of like fermentations Were conducted under varying conditions as illustrated in the following table. The assay results were obtained using the organism Streptococcus haemolyticus Strain C.-203 as a test organism with Difco-Bacto Brain-Heart Infusion reconstituted with Water and 11/2 percent agar asl the growth medium.

ployed as will be evident from the specilic examples which follow:

A preferred isolation procedure involves the steps of adsorbing the antibiotic from the filtered fermentation liquor onto activated carbon and thereafter eluting the activity with a polar solvent. The activity is adsorbed on the charcoal at the natural pH of the fermentation liquor which may be from about pH 6.5 to 8.0. The eluant is, preferably, a Water-miscible polar solvent, such as a 95 percent acetone-5 percent Water mixture. No particular adjustment of the hydrogen ion concentration of the eluant is necessary. Instead of aqueous acetone, we may use methanol acidified to a pH of 2 to 3 or at a pH as high Kas 9. Other alcohols such as ethyl alcohol, the propanols, the butanols, and the like, may also be used as eluants. These may be cut with water to percent or more when desired. Other hydroxylated, water miscible polar solvents include the Cellosolves, such as the ethyl ether of ethyleneglycol, and the like. Dilute acids such as twentieth normal hydrochloric acid and acetic acid may also be used as the eluant to recover the adsorbed antibiotic from the charcoal. Other polar solvents which may be used to recover the activity from the activated carbon include pyridine, aqueous solutions of phenol and still others of similar type.

The adsorption and elution may be carried out in various ways familiar to those skilled in the art. The filtered fermentation liquor may be simply stirred with the activated carbon and then filtered 'and the activity recovered from the lter cake by passing the eluant through the cake or by stirring it with theeluant. Another common procedure would be to pass the fermentation liquor through a column packed with activated carbon and thereafter pass the eluant through the column. Washing with Water to remove Water soluble impurities which are not strongly adsorbed on the activated carbon may precede -the elution.

After recovering the activity from the activated carbon, it is generally desirable to concentrate the eluant. If desired, the concentration may proceed to dryness and the material stored until ready for further purification. Because of the very high antitrypanosome activity of the Inoculum pH Capacity Aerationof Tank Llters of Temp., Ratio: Time .Assay Example in Medium Liters of Age of C. Vol. of Air in Hours Zone Gallons y Bottle Bottle to Vol. of H Widths culture culture Medium Farmen, P

in Hours tation *D 6. 96 I soo 1, soo 12 23 26-29 0.85 9g 2: 222* "-6 112. 5 6. 64 31. 2 *0 7. 02 0 6.72 II 100 200 6 28. 5 28 0. 95 48 6. 52 23. 3 72 7. 13 27. 7 89 7. 22 31. 3 p *o 7. ge m 10o 20o s 2e 2s o. Q5 74H2 gj ,g "5i- 91. 5 6. 70 32.7 *0 7. 18 0 6. 55 IV 200 400 6 30. 5 27-29 1. 00 72. 5 6.80 29 2 S9 7. 24 3o. 6

*Before sterilization. Without the asterisk, 0 indicates after sterilization.

ISOLATION PROCEDURES Several methods of recovering the antibiotic from the fermentation liquor, which depend upon the physical and chemical properties of the antibiotic, have been Worked out. In general, it is preferable to filter the fermentation liquor to remove the mycelia and other insoluble components of the fermentation by filtration at the pH of the harvest. A filter aid, such as diatomaceous earth, is employed. The antibiotic may then be adsorbed on an Iadsorbent and eluted therefrom. Solvent extraction,

material, it may be suitable for use in the treatment of animals at this state of the procedure. In fact, the antibiotic is so highly active that the fermentation liquor itself has been found to be effective -in vivo against Tryprznosoma equiperdum.

Further purification may be carried out by activated carbon chromatography. In this procedure a column of activated charcoal is prepared and the concentrated antibiotic dissolved in a suitable solvent such as 50 percent acetone-50 percent Water and passed through the column.

chromatography, and salting out procedures are also ern- 75 The column is then developed by Ithe continued passage of a similar solvent the weakly adsorbed impurities in the column are eluted and removed. When the antibiotic activity commencesto come through the column Ias determined by simpleaactivity tests of the type described herein elsewhere, a stronger eluant of the type described above is then passed though the carbon bed and the activity thus recovered.

The eluant may be concentrated or dried by lyophilization. Frequently, crystals of the antibiotic are recovered vas a result of the concentration step.

Y Further purification of the antibiotic may be achieved by several methods, some of Which will be illustrated hereinafter. A preferred method is the use of normal butanol and a partition column made up of a diatomaceous earth as illustrated in Example 9 It will be understood, of course, that these procedures are merely exemplary and are not meant to be taken as restricting the present invention to any particular method ofpurication. I

EXAMPLE V Preliminary purijcaton-Adsorption column The fermented liquor from several tanks as described above was pooled to give a mash volume of 1,450 liters at pH 7.1. The mash was ltered with the aid of Hyflo Super-Cel (diatomaceous earth) to give 1,300 liters of fil-trate. Assay indicated a total of 5.64 grams of pure antibiotic in the 1,300-liter solution. 1,950 grams of VDarco G-60 (activated charcoal) was added, and the cent water. y A total of 68.5 litersrof this y solution was usedv for Vdeveloping the column.` V- I The *rst 65 liters of percolate were collected, the lrst l0 liters-as one cut, and the next 55 liters as a second cut. 46 percent of the original 5,64 grams of activity. The first cut was concentrated under reduced pressure to an Vaqueous solution of 2 liters, `and the second cut was similarly concentrated to 3- 5 litersof aqueous solution. Each concentrate Ywas then separately lyophilized, yielding as follows; i

Cut No. 1-46 grams shown by assay to contain the equivalent of 0.94 gram crystalline antibiotic.

Cut No. 2.-59.'5 grams shown by assay to contain the equivalent of 1.92 grams crystalline antibiotic.

EXAMPLE VI Preliminary puriycaton.-Adsorpton-elution procedure Three hundred fty liters of mash at pH 7.4 was liltered with the aid of Hylio Super-Cel to give 300 liters of iltrate. The pH was adjusted to 6-7, and 600 grams of Darco G-60 was added. The mixture was stirred for one-half hour, about 1,200 grams of Hyo Super-Cel was added, and the slurry was stirred to homogeneity and filtered. The filter cake was slurried in 10 liters of water for 5 minutes and filtered. The washed cake'was slurried in a solution of 5,700 ml..of acetone and 300 ml. of water for 20 minutes and filtered. The volume of this rst These cuts contained respectively 17 percent andv eluate was 5.5 liters.

VEXAMPLE VIIY Preliminary purification-Solvent extraction The fermentation mash from tank 60 (prepared substantially as in Examples I-IV) was treated just as described in Example VI except that the final aqueous concentrate was not lyophilized. Thevolume of the concentrate was 2 liters, and this solution was adjusted to pH 2 (with hydrochloric acid) and extracted twice with 1 liter each time of n-butanol. The aqueousresidue was adjusted to pH 7 with'aqueous sodium hydroxide and saturated with about 1,200 `grams of ammonium sulfate. The resulting mixture was/,extracted three times with 1 liter each timerofbutanol. A certain amount of insoluble material which settle out at the interface during extraction was kept from the extracts. The following tabulation indicates the results of these extractions:

ventized, concentrated, and lyophilized vto give material which assayed 60 'y of activity per mg.

EXAMPLE VIII Further purification-Adsorption column Y An adsorption column was prepared Yas *follows-z 3,000

' grams of celite 545 and 1,000 grams o fparco C-,6Q'were mixed intimately in a pail tumbler. To this was'added 2,000 m1. ofv a 50 percent acetone-'5.0 percent Ywater'solution, and the whole was mixed thoroughly.-The-nal homogeneous mixture was packed with tampi'nginto an inverted 20.-liter bottle, the bottom of-ewhich had bcn c ut oi, to form a column 1l. inches in diameter and 13-14 inches high.

The percolate was fractionated as follows:

f Volume Total Total Fraction in liters solids in Activity grams Uni 8.5 7. 56 e. 9 1o. s 19. 4 1o 1o. 2 7. 7 79. 8X10 9. 4 a. s 31. 2 10 yThe last three fractions vwere desolventizedl under reduced pressure and lyophilized with the'following results:

The total recovery of activity units in the three dried samples was about 112 106 units. 1 'y of crystalline antibiotic=45 activity units.)

EXAMPLE IX Further purification- Partition column n-Butanol and water were stirred together in a separatory funnel and separated to give a pair of mutually saturated solutions. Approximately 500 ml. of n-butanol was added` to about 15 liters of butanol saturated with water, so that the solvent phase used in this column was butanol notV quite saturated with water.

One kilogram of acid-washed Celite 545 was intimately mixed with 500 ml. of aqueous phase, and the mixture was packed to form a column 42 sq. cm. in cross section and about 83 cm. high.

Samples from Examples V and VI Weighing 14.9 grams, and containing about 8,850 activity units per mg., were pooled and added to about 153 ml. of aqueous phase, and the mixture was stirred constantly at room temperature while the pH was brought to 2.0. After further stirring, the mixture was filtered to removeI a small amount of insoluble material. From the ltrate 3 ml. was removed for assay and total solids determinations, and the remaining 150 ml. was mixed with 300 grams of acid-washed CeliteV 545. The resulting mixture was packed on top of the column, adding thereto another 25 cm. to give a total height of 108 cm. The column was then developed with solvent phase, and the percolate was cut as follows :l f

Fractions F10, F11, and F12 were combined, concentrated under reduced pressure to desolventize, and lyophilized to give 3.34 grams of product.

EXAMPLE X Crystallzaton ofthe new antibiotic 3.34 grams of partially purified antibiotic was dissolved in 80 ml. of water at 65 C., and the pH of the solution was adjusted to 1.5 with dilute hydrochloric acid. The mixture was tiltered to remove a certain amount of insoluble gum. The pH of the iiltrate was adjusted to 4.0 with dilute aqueous sodium hydroxide, va seed crystal was added, and the mixture was left overnight at 4-5 C. The next morning the crystals were filtered, washed with cold water, and dried in a vacuum desiccator over Drierite at 1 mm. pressure for about 24 hours-yield, 1.19 grams.

Crystallizatin of the new antibiotic after butanol extraction of impurities A number of samples purilied 'through the rst adsorption such as the sample of Example VI were pooled and carried through the second adsorption column purication as described in Example VIII. The antibiotic rich portion of the percolate was concentrated under reduced pressure to a small aqueous volume and lyophilized as in Example VIII. A sample weighing 1.67 grams andcontaining a total of about 12.8 106 activity units was dissolved in ml. of water, and the solution was made 0.1 N lwith respect to hydrochloric acid by adding acid. This solution was extracted twice with 80 ml. each time of n-butanol, thereby removing about 1.07 grams of total solids, but only about 9 percent of the activity. The pH of the residue was adjusted to 6.5 and the resulting solution was desolventized under reduced pressure and then concentrated to 5-10 ml. and kept chilled. The crystalline product was iltered, redissolved in about 10 ml. of water at pH 3 -4 by warming slightly, and ltered. The filtrate was chilled, and the resulting crystals were filtered, washed with cold water, and driedyield, 145 mg. of white crystals.

EXAMPLE XII j Recrystallzation of and preparation of analytical samples 4.522 grams of various pooled lots of crystalline antibiotic (prepared as described in Example X) was dissolved in 200 ml.- of water at 60 C., and the pH- of the solution was adjusted to 2.5. The solution was filtered, and the filtrate was adjustedto pH 4 and left overnight at 4-5 C. VThe crystals were ltered and the same rrecrystallization procedure was repeated 4y times. kThe final recrystallization included a decolorizing step with a small amount of charcoal. The nal product was 1.89 grams (after drying at 2 mm. pressure). of analytical sample. Of this sample, 1.25 grams was further dried for two hours in abderhalden pistol at the boiling point of chloroform. The product obtained consisted of mats of thread-like (loform) crystals. The principal vibration directions and their v corresponding refractive indices were diicult to determine and are not given in view of the possibility of error.

1 EXAMPLE XIII Dextrin percent-- 1 N-Z Amine, A (hydrolyzelcasein) ..do 1 rNaClrln j 0.2 (NH4)2HPO4 d0 0.2 KH2PO4 do..-" 0.15 10-2-- MgSO4.7H2O do 0.025

Hoaglands Salt Solution percent (by vol.) Tap water to EXAMPLE XIV Equal volumes of n-butanol and the above beer were shaken together at the indicated pH values and then separated. The tabulation below shows the activities in the butanol extracts and aqueous residues With the extraction coe'icients.

wie.

13 i vEXAMPLE xv EXAMPLE XVI 11.9 liters of a similar beer was stirred at harvest pH with 55 gm. of Darco G-60 for one half hour. The suspension was filtered with the aid of a small amount of Celite and washed on a Buchner funnel with 500 ml. of water.

The washed cake was then slurried about twenty minutes with 300 ml. of acetone and filtered. The residual cake was re-eluted twice with 300 mls. each time of 95 percent acetone. The three acetone eluates were pooled and assayed, the results showing a recovery of 35 percent of the activity in the beer. The pooled solution was concentrated under vacuum to an aqueous concentrate which was lyophilized giving 3.64 gms. of solids, determined by assay to represent material purilied 11 fold compared to the antibiotic in the beer.

Beta methoxyethanol or -ethoxyethanol may be substituted for butanol or acetone in the above extraction.

EXAMPLE XVII 50 mg. of the antibiotic of Example XII, free base, was dissolved in about 5 ml. of water by the addition of 0.1 N hydrochloric acid, and 5 ml. of a saturated aqueous picric acid solution was added. The resulting crystalline precipitate was centrifuged and washed with about 5 ml. of cold water.

'I'he product was recrystallized three times from water with a hot filtration (6G-75 C.) in the final step, and dried for 24 hours at 1 mm. and finally over p205 at l mm. and 110 C. for 4 hours. Yield: 35 mg. of bright yellow crystals of the picrate salt.

Analyss.-Calcd for CNH18015N9S: C, 32.9; H, 2.91; N, 20.3; S, 5.17; O (dii), 38.1. Found: C, 33.19; H, 3.13; N, 20.19; S, 5.70; O (dii), 37.79.

Melting point 14S-144 C. (uncorr.) with slight decomposition.

Ultraviolet a max. (mp) 253 (20 ly/ml. EtOH).

EXAMPLE XVIII 150 mg. of the antibiotic, free base, was dissolved in 5 ml. of glacial acetic acid and treated with 5 ml. of glacial acetic acid saturated with hydrogen chloride.

The white precipitate of the hydrochloride salt which formed was removed by ltration, washed with l ml. of glacial acetic acid and 10 ml. of anhydrous ethyl ether, and dried for 24 hours over Drierite (calcium sulfate, anhydrons) at 1 mm. and room temperature.

EXAMPLE XIX A solution of 150 mg. of the free base in about 10 ml. of absolute ethanol was treated with a mixture of ml. of absolute ethanol and about 4 drops of concentrated sulfuric acid. The resulting precipitate of the sulfate salt was filtered, washed with absolute ethyl ether, and dried at 1 mm. for 24 hours. The product was amorphous.

14 EXAMPLEXX A saturated aqueous solution containing about mg. ef the free, basewas treated with a Vsaturated aqueous methyl Orange solution to yield, acolored. Crystalline PYB- cipitate ofthe helianthate salt.

We claim: n

1. Nucleocidin having the following structure;

where Ris the residue of a monose.

2. An anticiotic substance effective against trypanosomes and amoebae selected from the group consisting of a substance containing the elements, carbon, hydrogen, nitrogen, sulphur, and oxygen, in substantially the following proportions: carbon 34.0 percent; hydrogen 4.0 percent; nitrogen 21.6 percent, sulphur 8.3 percent; oxygen 32.0 percent, in the following structure:

in the temperature range of 17 C. to 42 C. for a pe-A riod of at least 48 hours at a hydrogen ion concentration within the range of about pH 6-pH 8.

4. A method of producing an antibiotic elective against trypanosomes and amoebae which comprises the steps of aerobically fermenting an aqueous nutrient medium with a microorganism of the species Streptomyces calvus within the temperature range of 17 C. to 42 C. for a period of at least 48 hours a-t a hydrogen ion concentration within the range of about pH 6-pH 8, thereafter contacting the water soluble components of the fermentation liquor with the adsorbent, separating the adsorbent from the liquid and recovering the antibiotic from said adsorbent by elution with a polar solvent.

5. A method of producing an antibiotic effective against trypanosomes and amoebae which comprises the steps of aerobically fermenting an aqueous nutrient medium with a microorganism of the species Streptomyces calvus within the temperature range of 17 C. to 42 C. for a period of at least 48 hours at a hydrogen ion concentration within the range of about pH G-pH 8, thereafter contacting the water soluble components of the fermentation liquor with activated charcoal, separating the charcoal from the liquid and recovering the antibiotic from said charcoal by elution with a polar solvent.

6. The process in accordance with claim 5 in which the solvent is a mixture of acetone and water.

(References on following page) References Cited fn the kfiley of this patent Thomas et al: Abstracts ofPapers 95 and 96 presented at 4th Annual Symposium on Antibiotics, Oct. 17-19, UNITEDYSTATESPATENTS *1956;:Washingtom D.C., 2 pages.- 1

Annals of the N.Y. Acad. of Sci., 60"(1954), page 5. Baker etal: I. Amer. Chem.y Soc.; Jan.; 5; .1955 (pp. Baldacci: Archives fur Mikrobiologie Bd. 2U, pp. 347- l-24). v L Y. I 357,1954. 11.0 

1. NUCLEOCIDIN HAVING THE FOLLOWING STRUCTURE:
 3. A METHOD OF PRODUCING AN ANTIBIOTIC EFFECTIVE AGAINST TRYPANOSOMES AND AMOEBAE WHICH COMPRISES THE STEP OF AEROBICALLY FERMENTING AN AQUEOUS NUTRIENT MEDIUM WITH A MICROORGANISM OF THE SPECIES STREPTOMYCES CLVUS WITH IN THE TEMPERATURE RANGE OF 17*C. TO 42*C. FOR A PERIOD OF AT LEAST 48 HOURS AT A HYDROGEN ION CONCENTRATION WITHIN THE RANGE OF ABOUT PH 6-PH8. 